Mass spectrometry is an analytical technique where ions of sample molecules are produced and analysed according to their mass-to-charge (m/z) ratios. The ions are produced by a variety of ionisation techniques, including electron impact, fast atom bombardment, electrospray ionisation and matrix-assisted laser desorption ionisation. Analysis by m/z is performed in analysers where the ions are either trapped for a period of time or fly through towards the ion detector. In the trapping analysers, such as quadrupole ion trap (Paul trap) and ion cyclotron resonance (ICR cell or Penning trap) analysers, the ions are spatially confined by a combination of magnetic, electrostatic or alternating electromagnetic fields for a period of time typically from about 0.1 to 10 seconds. In the transient-type analysers, such as magnetic, quadrupole and time-of-flight analysers, the residence time of ions is shorter, in the range of about 1 to 100 μs.
Tandem mass spectrometry is a general term for mass spectrometric methods where sample ions of desired mass-to-charge are selected and dissociated inside the mass spectrometer and the obtained fragment ions are analysed according to their mass-to-charge ratios. Dissociation of mass-selected ions can be performed either in a special cell between two m/z analysers, or, in trapping instruments, inside the trap. Tandem mass spectrometry can provide much more structural information on the sample molecules.
To fragment ions inside the mass spectrometer, collisionally-induced dissociation (CID) is most commonly employed. In the predominant technique, the m/z-selected ions collide with gas atoms or molecules, such as e.g. helium, argon or nitrogen, with subsequent conversion of the collisional energy into internal energy of the ions. Alternatively, ions may be irradiated by infrared photons (infrared multiphoton dissociation, IRMPD), which also leads to the increase of the internal energy. Ions with high internal energy undergo subsequent dissociation into fragments, one or more of which carry electric charge. The mass and the abundance of the fragment ions of a given kind provide information that can be used to characterise the molecular structure of the sample in question.
Both collisional and infrared dissociation techniques have serious drawbacks. Firstly, increase of the internal temperature causes intramolecular rearrangements that can lead to erroneous structure assignment, as discussed in Vachet, Bishop, Erickson and Glish, (1997) Am. Chem. Soc. 119: 5481-5488. Secondly, low-energy channels of fragmentation dominate, which can limit the multiplicity of cleaved bonds and thus the fragmentation-derived information, and in case of the presence of easily detachable groups result in the loss of information on their location. Finally, both collisional and infrared dissociations become ineffective for large molecular masses.
To at least partially overcome these problems, electron capture dissociation (ECD) has recently been proposed (see Zubarev, Kelleher and McLafferty (1998), J. Am. Chem. Soc. 120: 3265-3266).
The ECD technique is technically related but physically different from earlier work of using high-energy electrons to induce fragmentation by collisions with electrons (Electron Impact Dissociation, EID). U.S. Pat. No. 4,731,533 describes the use of high-energy electrons (about 600 eV) that are emitted radially on an ion beam to induce fragmentation. Similarly, U.S. Pat. No. 4,988,869 discloses the use of high-energy electron beams 100-500 eV, transverse to a sample ion beam to induce fragmentation. The method suffers though from low efficiency, with a maximum efficiency of total fragmentation of parent ions of about 5%.
In contrast to EID, in the ECD technique positive multiply-charged ions dissociate upon capture of low-energy (<1 eV) electrons in an ion cyclotron resonance cell. The low-energy electrons are produced by a heated filament. Electron capture can produce more structurally important cleavages than collisional and infrared dissociations. In polypeptides, for which mass spectrometry analysis is widely used, electron capture cleaves the N—Cα backbone bonds, while collisional and infrared excitation cleaves the amide backbone bonds (peptide bonds). Combination of these two different types of cleavages provides additional sequence information (Horn, Zubarev and McLafferty (2000), Proc. Natl. Acad. Sci. USA, 97: 10313-10317). Moreover, disulfide bonds inside the peptides that usually remain intact in collisional and infrared excitations, fragment specifically upon electron capture. Finally, some easily detachable groups remain attached to the fragments upon electron capture dissociation, which allows for determination of their positions.
The drawback of current electron capture dissociation methods lies in their relatively low efficiency, which manifests in the long time of electron irradiation. In order to obtain electron capture by a desired proportion of polypeptide parent ions, at least several seconds of irradiation is required for doubly-charged parent ions (see Zubarev et al. (2000) Anal. Chem. 72: 563-573). Typical parameters for the ECD technique are described in Zubarev (2000) ibid. Electron beams of 0.3-1 μA are used with average electron energy of about 0.5 or 1.0 eV. The higher currents are not found to provide more efficient ECD. It is stated that ECD requires a near-zero translational energy difference between the ions and electrons. When admitting different energy populations of electrons to the ICR cell, it is found that the lower energy electrons provide higher ECD efficiency.
This long irradiation time reduces the duty cycle of the mass spectrometer to 3-10%. In electrospray ionisation, sample ions are produced continuously and only a small fraction of these ions can be analysed in ECD experiments due to the poor duty cycle, resulting in low sensitivity. In addition, electron capture dissociation is an energetic process, resulting in scattering of the fragments. Insufficient collection of produced fragment ions additionally decreases the sensitivity. The long irradiation time makes electron capture dissociation possible only on ion cyclotron resonance m/z analysers that are among the most expensive types of mass spectrometers, and not in common use. Indeed, in transient analysers the residence time of ions is too short for effective electron capture. In Paul ion traps, the presence of alternating electromagnetic field of several hundred volts amplitude would rapidly deflect the beam or otherwise increase the kinetic energy of electrons above 1 eV, with the cross section for electron capture dropping by at least three orders of magnitude.
For these reasons, it would be desirable to shorten the ion-electron reaction and improve the efficiency of collection of fragments to make ECD more useful. It would be further highly desirable to allow the ECD technique to be used in other types of mass spectrometers.